Abstract

Subthreshold voltage dynamics are essential for neuronal information integration, yet they remain technically challenging to measure in vivo. While genetically encoded voltage indicators are emerging as powerful tools for voltage recording, they lack the sensitivity to detect millivolt-scale subthreshold fluctuations with two-photon microscopy—the method of choice for deep-tissue recording. To overcome this limitation, we engineered two genetically encoded voltage indicators, JEDI3sub and JEDI3hyp, with enhanced subthreshold voltage detection under two-photon excitation. In the mouse brain, JEDI3sub enabled simultaneous tracking of subthreshold optical tuning from over 100 cells simultaneously, while JEDI3hyp captured subthreshold dynamics associated with sharp-wave ripples in hippocampal PV⁺ interneurons. Moreover, JEDI3hyp supported prolonged imaging of brain-state-dependent, millivolt-scale subthreshold voltage changes across deep-layer somas, fine dendritic structures and diverse cell types. By enabling sensitive reporting of subthreshold voltage dynamics, JEDI3 indicators open previously unexplored avenues for dissecting neural information processing in health and disease.